It is well known that humans can regenerate T cells via thymic-dependent pathways or via thymic-independent homeostatic peripheral expansion. Thymic dependent pathways are superior in quality due to their capacity to regenerate a broad T cell receptor repertoire, but the reality is that very few humans who sustain T cell depletion have sufficient residual thymic function to restore T cells using this pathway. Thus, most patients rely on homeostatic peripheral expansion (HPE) to restore T cell populations and HPE typically results in chronically reduced CD4+ T cell numbers, inverted CD4/CD8 ratios and immune dysfunction. Our current paradigms for understanding T cell homeostasis and pathways of immune reconstitution provide no construct for understanding why humans have a profound inability to restore CD4+ T cells via thymic-independent HPE whereas they can readily restore CD8+ populations via HPE. A major accomplishment of this project during FY2008 was the completion of a series of studies that identified the factors limiting CD4+ immune reconstitution via HPE. Our previous work demonstrated that lymphopenia in humans results in elevated circulating interleukin-7 (IL-7) levels and current concepts held that IL-7 would serve to drive CD4+ HPE. However, based upon the fact that relationships between CD4+ counts and IL-7 in lymphopenic humans were strongly inverse, we hypothesized that chronic elevations in IL-7 might serve to diminish CD4+ HPE. To test this, we used mice genetically deficient in the recombinase gene (Rag-/-) to provide a milieu for lymphopenia, and investigated the biology of IL-7 in this setting more closely. We observed that lymphopenic mice also show elevated levels of IL-7 in their tissues but this is not due to increased production, but rather diminished utilization. Furthermore, despite the elevated IL-7 levels, CD4+ T cells do not efficiently expand in this milieu whereas CD8+ T cells do. The breakthrough observation came when we compared CD4+ homeostatic peripheral expansion in chimeras wherein IL-7 was either exclusively produced by bone marrow derived populations (and not produced by radioresistant host tissues) vs. chimeras wherein IL-7 was produced by radioresistant tissues and marrow derived populations or chimeras wherein IL-7 was produced by radioresistant tissues but not marrow derived populations. These studies provided the surprising result that IL-7 produced by bone marrow derived populations was sufficient to support CD4+ HPE but that the production of IL-7 by radioresistant tissues paradoxically diminished CD4+ HPE. Furthermore, we observed that elevated IL-7 levels that accumulate in lymphopenic mice (as a result of production by radioresistant tissues) induces downregulation of MHC Class II expression on antigen presenting cell (APCs) populations and diminished IL-7 production by the same APCs. Remarkably, if IL-7 signaling on APCs was inhibited by absence of IL-7 receptor alpha, lack of the common gamma chain receptor or by lack of STAT5, CD4+ HPE was greatly enhanced. This work therefore identified an entirely new regulatory axis for controlling CD4+ HPE, which primarily involves IL-7 signaling on APCs. This is paradigm changing since IL-7 signaling on APCs has not previously been shown to be of any real importance, yet this work implicates this axis as a fundamental regulator of CD4+ immune reactivity. This work has been submitted for publication and is currently under review. We plan to further examine the impact of this work by investigating whether manipulation of IL-7 signaling pathways on APCs could be enhance peripheral CD4+ niches and potentially enhance adoptive immunotherapy of CD4+ T cells or enhance CD4+ mediated antitumor immunity. We also will seek to modulate IL-7 signaling on APCs in the context of autoimmunity to determine whether this axis may be a therapeutic target for new immunosuppressive therapies. A second major accomplishment of work undertaken in this project during FY2008 has been the elucidation of the essential factors present during lymphopenia that augment the effectiveness of adoptive cell therapies. Many groups are currently subjecting patients to lymphocyte depletion as a means for improving the milieu within which adoptively transferred cells may proliferate and survive in vivo. While this appears to be effective, it brings with it the negative impact of lymphopenia, which can induce long term morbidity and can also limit the immune repertoire available to reject the tumor. Current concepts hold that the beneficial immune physiology of lymphopenia is due to increased homeostatic cytokines and diminished numbers of regulatory T cells. We therefore tested the hypothesis that provision of increased IL-7 and depletion of regulatory T cells would provide an equivalent or superior milieu for adoptive immunotherapy. We utilized the B16 melanoma model and adoptively transferred T cells with specificity for the immunodominant antigen on this tumor. We observed that adoptive immunotherapy administered to lymphopenic mice provided only a modest benefit compared to T cell replete mice however specific depletion of CD25+ T cells using a monoclonal antibody substantially improved the effectiveness of adoptive immunotherapy. This was further augmented in thymectomized hosts when it was combined with recombinant human IL-7. Such mice showed substantially improved survival following adoptive immunotherapy compared to lymphopenic mice. The inferior milieu of the lymphopenic mouse was due to two major factors. First, while adoptively transferred T cells proliferated more rapidly in the lymphopenic milieu compared to mice treated with regulatory T cell depletion and rhIL7, this did not result in efficient accumulation presumably due to a higher rate of programmed cell death and did not result in potent effector activity as measured by cytokine production. Secondly, the non-lymphopenic hosts showed evidence for epitope spreading which improved the effectiveness of the adoptive therapy which was lacking in the lymphopenic hosts. We therefore conclude that lymphopenia should not be considered a requisite setting for adoptive immunotherapy but that targeted approaches that provide the specific components of the altered physiology of lymphopenia are preferable and will be more effective in the long run. This work is currently in preparation for submission for publication. Other accomplishments from this project include a collaborative report which demonstrated for the first time that IL-7 upregulates Fas expression on T cells and predisposes these cells to programmed cell death (Fluur et al, J Immunol 2007; 178, 5340). This observations corroborates previous work published by our group demonstrating that T cells from lymphopenic hosts show an increased rate of programmed cell death. We contributed to this work by providing data from previously conduced rhesus macaque studies wherein IL-7 administration induced Fas upregulation and also by investigating a relationship between IL-7 levels during lymphopenia in humans and Fas expression. This work utilized cryopreserved T cells from lymphopenic hosts collected on NCI IRB approved trials as part of our clinical program. Work from this project during FY2008 also demonstrated that the thymopo [summary truncated at 7800 characters]